Heterochromatin is a genomic structure that functions in all eukaryotes to determine cell identity and maintain genome stability by silencing genes. The requirements for heterochromatin assembly are incorporation into chromatin of silencing protein complexes with histone modification activity that leads to repressed gene expression. This general mechanism is conserved in all eukaryotes, though multiple varieties of heterochromatic domains exist, even within the same organism. Much is known about the factors that contribute to heterochromatin, yet many key events in specific pathways are poorly understood. One challenge in studying heterochromatin is the fact that manipulation of the central components in vivo often affects multiple types of heterochromatin or other related pathways in the cell. The aims of this project will address this problem in three ways: firstly, by using a functional in vitro reconstituted heterochromatin system to better understand the mechanism of heterochromatic silencing in an isolated environment. Secondly, using a pre- assembled heterochromatin domain to identify new factors within the cell that contribute to heterochromatin formation and maintenance. Third, by establishing a system to address the contribution of specific non-coding RNA elements to initiation and maintenance of heterochromatin. Initial studies in yeast models of heterochromatin will make important contributions to the field of chromatin biology and will also serve as a model for subsequent studies in the human system to be carried out after the transition to independent investigator. The primary goal of the project is to comprehensively characterize heterochromatin domains in order to compare conserved features that are central to gene silencing and genome stability. The diversity of human heterochromatic domains is evident, yet the general principles that govern assembly of these domains remain quite similar to those in the budding and fission yeast models. These basic similarities make it feasible to compare results of yeast studies to humans and also to use the approaches that have been developed in the simpler yeast models as a guide to study human heterochromatin. This theme is the foundation of this proposal and the direction I will take as I transition to independence. PUBLIC HEALTH RELEVANCE: Heterochromatin is a genomic structure that functions in all eukaryotes to determine cell identity and maintain genome stability by silencing genes. The general requirements for heterochromatin assembly are the incorporation into chromatin of silencing protein complexes with histone modification activity that leads to repressed gene expression. Multiple varieties of heterochromatic domains exist within the same eukaryotic organism.